This invention relates to the isolation and purification of biologically active substances from thymus tissue.
A prerequisite for establishing that a mammalian organ has an endocrine function is a demonstration that cell-free extracts of the organ will replace specific biological functions of the organ. It is common knowledge that this criterion has been met for the thymus gland, because extracts from the thymus gland have been shown to restore deficiencies of immune function which are brought about by thymectomizing an animal, i.e., removal of the thymus gland. Consequently, it is believed that a hormone or hormones unique to the thymus gland control immune competence in man.
It is also common knowledge that there is a direct correlation between certain diseases of childhood, aging or senescence and thymus-dependent immunity. It is known that the thymus has already begun to atrophy when children are about 10 years in age, and that the decrease in size continues so that the size has decreased to a level of about 50% by 50 years, and that the size continues to diminish with advancing age. Common diseases related to aging include cancer, autoimmune diseases and infectious diseases.
Various investigators have proposed that a hormone or hormones of the thymus gland are peptides. Purified preparations, which are mixtures, have been obtained from thymus extracts, and peptides have been isolated which have diverse activities in biological assay systems which are relevant to the human immune system.
Miller, J. R. A. P. and Osoba, D., Ciba Found. Study Group 16, 62 (1963), provided direct evidence that the thymus produces a humoral factor which may be important to the control of immunity. The evidence was based upon the discovery that deficient immune responses in mice after neonatal thymectomy were prevented by the implantation of graphs of thymus tissue. In 1965, Osoba, D., J. Exp. Med. 122, 633 (1965), had the concept that thymus lymphocytes are functionally immature in situ, but when these lymphocytes leave the thymus, they are obtained immunological competence through maturation under the influence of a "competence-inducing factor" or hormone produced by the thymus. Osoba found that this "competence-inducing factor" was in the thymus and was not in the spleen or lymph nodes. These and studies by other investigators were largely physiological, and provided a basis for the entrance of new chemistry of fractionation towards the isolation of one or more such thymic hormones.
Abraham White was one of the Early investigators in the research to achieve purification by stepwise fractionation toward a factor or hormone. Allan Goldstein was one of his students in the research which started about 1965 and continued during subsequent years.
In 1975, Goldstein, A., standardized a purification through about five steps and designated the material as "fraction 5". They used this fraction 5 for diversified biological research and provided such fraction to other investigators and also for exploratory clinical studies in patients with immune deficiency diseases.
Fraction 5 has been important to study effectiveness of constituents in the thymus for the clinical treatment of thymus-dependent immunological diseases (Wara, D. W., Goldstein, A. L., Doyle, W. and Amman, A. J., New Engl. J. Med. 292, 70-74 (1975); Goldstein, A. L., Cohen, G. H., Rossio, J. K., Thurman, G. B., Brown, C. N. and Ulrich, J. T., Med. Clin. N. Amer. 60, 195-606 (1976)). Fraction 5 has also been reported to cause reversal of certain immunological parameters which are known to decrease in cancer patients (Goldstein, A. L., Cohen, G. H. and Thurman, G. B., Control of Neoplasia by Modulation of the Immune System, 241-253 (M. A. Chirigos, ed., Raven Press, NY) (1977)). When fraction 5 was used in a combined modality ot treat carcinoma of the lung by a randomized protocol, both survival time and number increased (Cohen, M. H., Chretien, P. B., Hide, D. C., Fossieck, B. E., Blum, P. A., Kenany, D. E., Lipson, S. D. and Minna, J. D., Proc. Amer. Assoc. for Cancer Res. 17, 117 (1978 )).
Kook, A. I., Yakir, Y. and Trainin, N., Cell Immunol. 19, 151-157 (1975), described their purification and partial chemical characterization of their thymic humoral factor (THF). Preparations of their THF were used by Varsano et al. (Acta Paediatr. Scand. 66, 329 (1977)) to treat severe disseminated adenovirus infection, which was reported in 1977 to be successful. A preparation of THF was also used by Zaizov et al. (Biomedicine, 27, 105 (1977)) for therapy with immunosuppressed children who had lymphoproliferative neoplasia and generalized varicella.
These exemplary clinical studies with preparations from A. Goldstein et al. and Trainin et al. underscore the importance of the chemical isolation and elucidation of thymic factors and hormones.
A summary of purified preparations and isolated peptides from the thymus, particularly as they have been tested for cyclic nucleotide effects, has been made by Naylor and A. Goldstein (Life Sci. 25, 301-310 (1979)). Supplementing this summary of 1979, Low and A. Goldstein et al. announced in 1980 (Fourth Intl Congress of Immunology, Abstract No. 17.2.22.) that they have isolated two new thymic peptide, designated thymosin .beta..sub.3 and .beta..sub.4, which appear to act on stem cells. These two peptides have an MW of about 5000-5500.
In 1977, Goldstein, A., et al. (Proc. Nat'l. Acad. Sci. 24, 725-729) reported on the isolation and sequence of thymosin .alpha..sub.1 from fraction 5, see also related U.S. Pat. No. 4,079,127.
Thymosin .alpha..sub.1 is a peptide having 28 amino acids, and it has AcSer in the N-terminal position. The sequence is as follows.
AcSer-Asp-Ala-Ala-Val.sup.5 -Asp-Thr-Ser-Ser-Glu.sup.10 -Ile-Thr-Thr-Lys-Asp.sup.15 -Leu-Lys-Glu-Lys-Lys.sup.20 -Glu-Val-Val-Glu-Glu.sup.25 -Ala-Glu-Asn.sup.28 -OH PA0 NH.sub.2 -Ser-Glu-Phe-Leu-Glu.sup.5 -Asp-Pro-Ser-Val-Leu.sup.10 -Thr-Lys-Glu-Lys-Leu.sup.15 -Lys-Ser-Glu-Leu-Val.sup.20 -Ala-Asn-Val-Thr.sup.25 -Leu-Pro-Ala-Gly-Glu.sup.30 -Gln-Arg-Lys-Asp-Val.sup.35 -Tyr-Val-Gln-Leu-Tyr.sup.40 -Leu-Gln-Thr-Leu-Thr.sup.45 -Ala-Val-Lys-Arg.sup.49 -COOH PA0 THYMOPOIETIN I: Gly.sup.1 Gln.sup.2 His.sup.43 PA0 (H) Arg.sup.32 -Lys.sup.33 -Asp.sup.34 -Val.sup.35 -Tyr.sup.36 -OH PA0 &lt;Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH
Synthetic thymosin .alpha..sub.1 is an ongoing subject of diversified biological and clinical investigations.
Gideon Goldstein and his associates began their research upon the thymus about 1969, and have continued this research up to the present time.
In 1974, Goldstein, G., reported on the isolation of thymin from bovine tissue. Thymin was found to consist of two polypeptides which were designated thymin I and thymin II. These two peptides were found to induce marrow cells to develop as intrathymic lymphocytes.
In 1975, Schlesinger and Goldstein (Cell 5, 361-365) reported upon the sequence of thymopoietin II. Goldstein and his associates renamed their two polypeptides as thymopoietin I and II. The expressions thymin I and I were abandoned.
Thymopoietin II has the following sequence:
Thymopoietin II differs from thymosin .alpha..sub.1 by having more amino acids, but the two peptides do have serine in a position 1 although Ser.sup.1 in thymopoietin II is not acetylated. Both thymopoietin II and thymosin .alpha..sub.1 have free C-terminal carboxyl groups.
Thymopoietin II differs from thymopoietin II only as follows:
Weksler, M. E., Innes, J. B. And Goldstein, G. (J. Exp. Med. 148, 996 (1978)) reported in 1978 some immunological studies on aging by utilizing mice. They observed two immune responses which are related to the thymus and which are affected by aging. One response concerned the generation of IgG plaque-forming cells (PFC). The second response concerned the generation of PFC with high affinity for antigen. They found that thymectomy of the mice accelerated both of these two immune responses. They also found that a preparation of thymopoietin increased the immune function of the spleen cells of old mice.
Since thymopoietins I and II are peptides having a relatively large number of amino acids, forty-nine, they designed and synthesized smaller peptides on the basis that such small peptides might simulate the activities of the native peptide. On this basis, Goldstein, G., et al. (Science 204, 1309 (1979)) synthesized a pentapeptide which was found to have a biological activity that was characteristic of the thymic hormone, thymopoietin.
The synthetic pentapeptide has the sequence:
This pentapeptide induced, in vitro, differentiation of murine prothymocytes to thymocytes. This pentapeptide also inhibited, in vitro, the differential induction of cells which had a B-lineage. These investigators concluded that these two activities of the pentapeptide are unique to thymopoietin.
Goldstein, G., et al. concluded that this synthetic pentapeptide may represent an active site of thymopoietin.
Nathan Trainin and his co-workers at the Weizmann Institute of Science in Israel initiated their study on a factor in the thymus about 1960, and Trainin and his successive associates have continued their studies to the present time. The basis of their study was the concept of the possible existence of a thymic humoral factor, which they designated THF, in due time.
In 1973, Trainin et al. reported upon the hormone-like activity of the THF which induced immune reactivity.
In 1974, Trainin et al. described data indicating that THF can induce immunocompetence through cAMp and DNA syntheses. During the period of 1975-6, Kook, A. I., Yakir, Y. and Trainin, N. (Adv. Exp. Med. Biol. 66, 215-220 (1976)) described chemical characterization data indicating that THF can be an acidic peptide having a molecular weight of ca. 3000 and which consists of ca. 31 amino acids.
Two amino acid compositions of preparations of THF were published in 1975 and 1976 as follows:
______________________________________ 1975 1976 ______________________________________ 4 3 Asp 1 2 Thr 5 3 Ser 8 5 Glu 2 1 Pro 5 3 Gly 2 2 Ala -- 1 Val 1 2 Leu -- 1 Phe 1 1 Lys 2 2 Arg -- 1 Cys 31 27 ______________________________________
In 1979, Rotter, V. and Trainin, N. (J. Immunol. 122, 414-420 (1979)) described a thymic plasma recirculating factor (TPRF). This factor was partially characterized as a peptide having a molecular weight of about 3000. Their biological data indicated that this factor may involved in T-cell maturation.
Alberto Astaldi, of the Central Laboratory of the Netherlands, appeared to have begun his investigations on the thymus about 1970. With his associates, Astaldi has published a series of interesting investigations up to the present time which are important, because the molecular weight of his apparent peptide appears to be lower than that of any other investigator in the field.
In 1978, Kruisbeek et al. (Cell. Immunol. 35, 145-147 (1978)) described a thymic epithelial culture supernatant (TES). They found that TES increased the antibody production to SRBC by spleen cells from athymic mice. Also, TES was found to induce a helper cell function in thymocytes.
In 1979, Wijermans et al. (Biochem. Biophys. Res. Commun. 86, 88-96 (1979)) described data showing the presence of a thymus-dependent factor in serum (SR). It was found by this group that SF increased cAMP levels and stimulated protein synthesis. Their data also indicated that SF is thymus-dependent.
In 1979, at the Miles International Symposium at Johns Hopkins University in July, Astaldi reported data on the chemical characterization of SF.
SF was apparently pure by the presence of "one spot" in TLC systems. It was estimated to have a molecular weight of less than 500 and probably to consist of four amino acids.
Jean Francois Bach and his associates appear to have initiated their investigations of the thymus about 1969, and have continued to make important contributions in this field up to the present time. During the early years, their investigations focused upon biological aspects of the thymus immunosuppression. Later, they undertook chemical purification studies on a factor.
In 1973, Bach et al. reported the demonstration of a circulating thymic hormone which they observed to be in the serum of normal mice, but not in the serum of athymic mice. This factor appeared to have a molecular weight of less than 10,000 on the basis of its passage through a membrane.
In 1977, Bach et al. described the isolation of a thymic peptide (FTS) from pig serum. They utilized a total of about 1000 liters of serum to achieve isolation of the peptide by steps including ultrafiltration, gel filtration and ion exchange chromatography.
FTS was found to be a nonapeptide which consisted of 1 Lys, 1 Asp(Asn), 2 Ser, 2 Glu(Gln), 2 Gly, 1 Ala.
Bach et al. (Nature 266, 55-57 (1977)) further characterized FTS in 1977, and described the sequence as:
It can readily be appreciated from the foregoing description that the thymus organ is a source for various hormonal substances, not all of which have presently been identified. Consequently, there is much need for the purification, isolation and identification of the discrete components which are present in thymus extracts. Unless the components are purified to homogeneity, there is no suitable procedure available to elucidate the chemical structure and to ultimately synthesize these biologically valuable hormones for clinical studies.